The detection of small amounts of peroxidatively active substances such as occult blood, hemoglobin, myoglobin, etc., in body fluids and in body excreta has long been recognized as an invaluable aid to the medical practitioner in the diagnosis of many abnormal conditions. For example, blood found in urine, feces or vomit is important for the diagnosis of hemorrhages in the stomach, intestines and urinary tract. Such hemorrhages are caused, for example, by tumors, ulcers and inflammations of the corresponding organs. In urine, the presence of peroxidatively active substances may be indicative of such abnormal conditions as typhus, scurvy, purpura, pyemia, nephritis, third degree burns, carcinogenic conditions, disease and infection of the urinary system, hemolytic toxins and post cardiac infarct.
It has long been recognized that in the presence of a hydroperoxide a chromogen can be oxidized to a colored substance and thereby indicate the presence of a peroxidate-active substance. This reaction has been used for quite a long time in medicinal and forensic analysis, especially for the detection of blood. Typically hydrogen peroxide is employed as the hydroperoxide and as a chromogen there is preferably used benzidine, ortho-tolidine or leuko malachite green. Examples of various procedures, compositions and devices described in the literature for detection of occult peroxidatively active substances appear in U.S. Pat. Nos. 2,290,436; 2,799,660; 2,838,377; 3,012,976; 3,092,463 and 3,092,464, all assigned to the present assignee.
While the compositions mentioned in the referenced patents provide rapid means for the detection of occult blood, the compositions are relatively insensitive to especially minute quantities of peroxidatively active substances corresponding to blood dilutions of less than about 1:20,000, i.e., about 200 to 300 intact red blood cells per microliter of sample (RBC .mu./l). In U.S. Pat. No. 3,290,117 the sensitivity of occult blood test compositions was shown to be markedly improved by the addition of quinoline or certain quinoline derivatives such as quinine. With the addition of quinoline derivatives to prior art occult blood compositions it was demonstrated to be possible to detect 5 to 50 .mu./l of sample corresponding to blood dilutions as low as 1:1,000,000. Similar sensitivities were obtained in U.S. Pat. No. 3,853,472 which discloses the use of fused polycyclic derivaties of quinoline as potentiating or activating agents. In addition, improved test compositions, devices and methods for detecting peroxidatively active substances are disclosed in U.S. Pat. No. 3,986,833. The latter patent makes specific reference to potentiating agents comprising acid salts or adducts of quinoline compounds.
Since the sensitivity of occult blood tests is of such great importance, it is essential that such tests not only be highly sensitive to peroxidatively active substances, but also retain their sensitivity. Unfortunately, except for the improved compositions containing quinine or certain other quinoline derivatives referred to in the prior art, many of the highly sensitive compositions which have been disclosed are unstable due to the volatility of added potentiators at room temperature or slightly above. Furthermore, virtually all of the prior art compositions utilize potentiators which are water insoluble and thus require suspension in organic solvents prior to incorporation into a test composition. It is common commercial practice for known potentiators to be suspended in an organic solvent solution containing the indicators. As a result, the indicators are often rapidly discolored in the presence of these potentiators and must be discarded. A desideratum of the art has been to provide test compositions which are not only rapid and highly sensitive, but which are also capable of retaining their sensitivity without discoloring the indicator system.